Macrophages are derived from circulating monocytes that exit the vasculature and invade into the surrounding tissues where they differentiate under the influence of local signals into resident tissue macrophages. Resident macrophages have a variety of roles; they patrol tissues for damaged or apoptotic cells, which they clear by phagocytosis, they identify and eliminate such invading pathogens as bacteria, fungi, and virally infected cells, they scavenge lipoproteins, and they are also responsible for regulating tissue oxygenation by influencing the formation of new blood vessels and modulating vascular permeability.
Macrophages are a highly versatile cell type with an impressive repertoire of functions depending on their location and activation status. This includes antigen presentation, anti-bacterial and antitumor activity, and the secretion of a wide variety of regulatory peptide factors, prostanoids, and enzymes.
Macrophages are therefore a population of immune cells that orchestrate a diverse array of functions including inflammation, tissue repair, and immune responses. This functional diversity is achieved by the remarkable heterogeneity of macrophages, which have the capacity to dramatically change their phenotype as a result of differentiated plasticity as well as local environmental cues.
As immune effector cells, the role of macrophages in inflammation and host defense is well characterized. Additionally, macrophages are integral in the promotion of proper wound healing as well as the resolution of inflammation in response to pathogenic challenge or tissue damage (Christopher J. Ferrante and Samuel Joseph Leibovich, 2012, Advances in wound care, 1: 1, 10-16). These diverse physiological functions stem from the remarkable plasticity of macrophages, which allows these cells to dramatically change their form and function in response to local environmental signals. Unstimulated macrophages are typically quiescent; stimulation of these cells, however, results in the development of markedly polarized phenotypes in response to molecular cues residing in the local microenvironment.
Current classification of macrophages recognizes polarization into two distinct phenotypes. Thus, macrophages are generally classified as either classically (M1) or alternatively (M2) activated. M1 macrophages have a proinflammatory phenotype exhibiting increased phagocytic activity and secretion of proinflammatory cytokines that aid in the removal of pathogens and abnormal or damaged tissues. M2 macrophages have a polar opposite phenotype exhibiting high levels of anti-inflammatory cytokines and fibrogenic and angiogenic factors that serve to resolve inflammation and promote wound healing (Martinez F O, Helming L, and Gordon S. 2009. Annu Rev Immunol; 27: 451-483). Both M1 and M2 macrophages express distinct molecular markers.
M1 macrophages are induced by recognition of pathogen-associated molecular patterns, such as bacterial lipopolysaccharides (LPS) and peptidoglycan, or damage-associated molecular patterns, such as released intracellular proteins and nucleic acids, as well as stimulation by the T-cell-secreted cytokine interferon gamma (IFN-γ). M1 represent a proinflammatory phenotype, exhibiting increased phagocytic and antigen processing activity as well as increased production of proinflammatory cytokines (e.g., interleukin 1 [IL-1], IL-6, IL-12, and tumor necrosis factor alpha [TNF-α]) and oxidative metabolites (e.g., nitric oxide and superoxide) to promote host defense and removal of damaged tissue. In contrast, M2 macrophages are induced by a variety of stimuli (e.g., IL-4/IL-13, glucocorticoids) and represent a phenotype that is potentially important in the promotion of wound healing and tissue remodeling as well as the resolution of inflammation (Martinez F O, Sica A, Mantovani A, and Locati M. 2008. Front Biosci; 13: 453-461).
The remarkable plasticity of macrophages has important implications for clinical science. Proper macrophage polarization is necessary in several important physiological processes including, but not limited to, wound healing, immune response, and nerve/muscle regeneration (Kigerl K A, et al., 2009, J Neurosci; 29: 13435-13444). Thus, it is not surprising that aberrations in macrophage polarization are associated with some of the pathology observed in defective wound healing, diabetes, muscular dystrophy, fibroproliferative diseases such as rheumatoid arthritis and liver and lung fibrosis, as well as tumor progression. Elucidating the specific microenvironmental signals that contribute to macrophage polarization could potentially lead to methods for the pharmacological manipulation of macrophage phenotypes to promote favorable processes (e.g., wound healing) or inhibit pathologic processes (e.g., fibroproliferative diseases and tumor growth).
As mentioned before, the ability of macrophages to alter their phenotype in response to different environmental stimuli has led to considerable research to both identify the wide variety of signals that induce these phenotypes as well as characterize the molecular profiles of M1 and M2 macrophages. However, macrophage polarization is a complex process and it has emerged that there is a broad set of signals that induce distinct macrophage phenotypes.
Of the increased numbers of macrophages present in diseased tissues, many are seen to accumulate in or adjacent to poorly vascularized, hypoxic sites, where considerable tissue damage may have occurred. High macrophage numbers have been reported in avascular and necrotic sites in breast and ovarian carcinomas, hypoxic areas of dermal wounds (Hunt, T. K., et al., 1983, Surgery, 96, 48-54), avascular locations of atherosclerotic plaques, the synovium in joints with rheumatoid arthritis, ischemic sites in proliferative retinopathy, and around vascular occlusions in cerebral malaria.
Macrophages are able to function under such adverse conditions by altering gene expression and adapting their metabolic activity. Hypoxia can induce marked changes in the secretory activity of macrophages, eliciting the release of both pro-angiogenic and inflammatory cytokines by macrophages in vitro and in vivo. Some studies have reviewed the effects of experimental hypoxia on various macrophage functions (Claire Lewis, et al., 1999, Journal of Leukocyte Biology, 66, 889-900; Maria M Escribese, et al., 2012, Immunobiology, 217, 1233-1240).
However, as hypoxia is usually transient in diseased tissues, and rarely, if ever, acts on cells in isolation from other pathogenic stimuli, these studies have highlighted the requirement of co-stimuli for its effects on macrophages.
On the other hand, methods based on physiotherapy for treating musculoskeletal injuries, including injuries to muscle, tendon and fascial components, immobilization, and various physiotherapies, have limited success. The use of drugs to reduce the pain is also commonly used, but none of these methods have been able to accelerate the healing process and reduce scar formation. The use of platelet rich plasma and stem cells seems to be a promising method for the treatment of these injuries, and several approaches has been developed, but none of these methods has achieved a complete regeneration of the injured tissue. Furthermore, the use of stem cell therapy has intrinsic risks given the loss of control on the administered cells once they have been implanted in the recipient with potential tumorigenic risk or inappropriate differentiation. Thus, improved methods of treating injured tissues in patients are required.
Ischemia preconditioning has been commonly used to prevent ischemia reperfusion injures. US2013184745, for example, describes a device of ischemic preconditioning. Several patent documents describe the use of blood flow restriction in muscle training (for instance, US2016235965 and US2016317864). But the effect of this technique has not been previously used in treatment of musculoskeletal injuries in order to accelerate the wound healing and tissue regeneration, reducing scar formation.
From the state of the art a plurality of medical devices for inducing hypoxia in cell culture are known. For example, in GB2499372 it is described a hypoxic pressure chamber for cell culture that comprises a plastic chamber with a gas inlet and a gas outlet. In U.S. Pat. No. 3,886,047 it is disclosed a hypoxic chamber with inlet and outlet gas tubes for culture growth under controlled atmosphere.
It is also known a culture chamber, which is described in US2003092178, where oxygen concentration is monitored to control the gas profile inside the chamber. Additionally, document WO2010058898 proposes a device comprising a culture chamber for cell culture, a vacuum pump for discharging air from the culture chamber to the outside, an automatic control valve for controlling a supply pipe of a carbon dioxide tank for supplying carbon dioxide into the culture chamber and a supply pipe of a nitrogen tank for supplying nitrogen into the culture chamber.
All these devices have in common that have been designed to culture cells on a hypoxic chamber and are useful for investigational purposes. None of these devices would be useful for clinical application since the processes are not carried out in aseptic conditions. In all the systems previously described, the cell cultures are placed and removed from the hypoxic chamber to external environment, and therefore can be easily contaminated.
On the other hand, several systems have been designed to separate and concentrate blood components from a blood sample. Some examples are patents ES1059764, U.S. Pat. No. 7,976,796 and US2016015884 but there are not systems described to induce hypoxia on the cells contained in these devices.
In summary, methods for obtaining macrophages polarized to a phenotype that promotes wound healing and, in general, tissue regeneration, are required. Specifically, methods for the artificial polarization of macrophages into an M2 phenotype would be of special interest, as M2 macrophages present a phenotype that is important in the promotion of wound healing and tissue remodeling as well as in the resolution of inflammation. The development of these methods would therefore enable the artificial manipulation of macrophage polarization to obtain macrophages phenotypes which enhance normal physiological processes, such as wound repair.